In this potentially high-reward R21 proposal, we explore the novel application of lipid-shelled, gas-filled microbubbles (used clinically as ultrasound contrast agents) as a method for creating cell laden microporous hydogels for cartilage tissue engineering. Rather than classical techniques of porogen leaching (which are often toxic), microbubble dissolution can be triggered "on-demand" by applied hydrostatic pressure or ultrasound. The latter affords unique spatial control of micropore formation in the hydrogel, which we anticipate will promote culture development of mechanically functional, large (anatomically-shaped) engineered cartilage constructs to serve ultimately as clinical alternatives to large allografts or joint implants. The microbubble dissolution process generates micropores that are homogeneously distributed within the gels, while enabling the direct ab initio immobilization of cels within the gels. Importantly, preliminary data demonstrates a 2-fold increase in mechanical properties of chondrocyte-seeded hydrogels with microbubble- derived microporosity versus control gels. This effect is greater than we have observed with applied deformational loading using chondrogenic media. We also have evidence that microbubbles promote more homogeneous axial properties. The proposed research to fabricate patella constructs is guided by these Hypotheses (H) &Specific Aims (SA): H1: Microbubble-infused hydrogel scaffolds exhibit increasing solute permeability in a microbubble dose- dependent manner. SA1. Fabricate chondrocyte-seeded hydrogel constructs with microbubble concentrations yielding initially 25%, 50%, 100% greater permeability of transforming growth factor beta 3 (TGF-23), a critical chemical factor in engineering of functional cartilage, than the hydrogel without microbubbles (0%). Measure solute permeability (P) and material properties including Young's modulus (EY) and dynamic modulus (G*). H2: Chondrocyte-seeded, hydrogel scaffolds incorporated with microbubbles will yield engineered tissues with properties closer to the native tissue compared to the same scaffolds without microbubbles. H2a. The properties of constructs with microbubbles are dependent on timing of microbubble dissolution. H2b. Application of applied dynamic deformational loading enhances the beneficial effects of microbubble-infused hydrogels. SA2a. Using microbubble conditions of SA1 (25%, 50%, 100% increase in TGF-23 permeability), culture constructs for 56 days with triggered dissolution of gas-filled microbubbles on day 0 or day 14. Measure material and biochemical properties, solute permeability, and perform histology on day 0, 14, 28 and 56. SA2b. Repeat SA2a using the best responding groups for microbubbles triggered on day 0 and day 14, but with application of daily dynamic deformational loading (10% deformation at 1 Hz, 3 hours/day). PUBLIC HEALTH RELEVANCE: An estimated 27 million Americans age 25 and older have osteoarthritis (OA), with the total direct cost of OA is estimated at $28.6 billion dollars a year in related medical costs. Effective treatment of cartilage injuries using tissue engineering strategies may prevent the development of OA and may reduce the need for a total joint replacement. In this proposal, the novel application of microbubble technology as a means of fabricating cell seeded hydrogel scaffold constructs with microporosity for cartilage tissue engineering is investigated.